3.4 Stability studies of PEG-FGF2 conjugates
To verify whether site-specific PEGylation improves the stability of
FGF2 conjugates, SD rat plasma and mice wound fluid were used.
Compounds 5 , 6, and 7 were first incubated
with rat plasma over 24 h and compared with FGF2 incubation. Solid-phase
extraction was conducted at different time points, and the remaining
FGF2 was analyzed using ELISA Kit to examine plasma stability
(Figure 5A ). The results showed rapid degradation of native
FGF2 in plasma, with its concentration decreased to less than 10% of
the initial value within 30 min and complete degradation within 1 h at
37 °C. In contrast, all PEG-FGF2 conjugates showed enhanced stabilities
relative to the unmodified FGF2. After 2 h of incubation in plasma, the
residual FGF2 percentages of compounds 5 , 6, and7 were about 66%, 93% and 90% of the initial concentrations,
respectively. After 8 h of incubation, about 33%, 76%, and 58% of the
initial concentrations remained for compounds 5 , 6,and 7 , respectively. The data indicate that site-specific
PEGylation can significantly improve the stability of FGF2 in plasma.
Acute wound fluid is generally rich in metalloproteinases (MMPs),
especially MMP2 and MMP9 subtypes, within the initial 3 days after scald
(Xu et al. , 2018), which may result in elevated levels of
proteases that can rapidly degrade exogenous growth factors (Ladwiget al. , 2002). Therefore, the stability of PEG-FGF2 conjugates
was further quantitatively analyzed by ELISA Kit after incubation in
wound fluids collected within 1 day after scald (Figure 5B ). As
expected, FGF2 mixed with wound fluid was degraded rapidly, for a
remaining FGF2 rate of 4.47 ± 0.95% after incubation in wound fluid for
24 h. Compounds 5 , 6, and 7 were more stable,
with remaining FGF2 rates of 32.00 ± 7.94%, 63.33 ± 3.51%, and 49.33 ±
5.13% respectively. These results indicate that all PEG-FGF2 conjugates
exhibit obviously increased stability in wound fluids compared to
unmodified FGF2.
Taken together, these results demonstrate that site-specific PEGylation
can effectively protect FGF2 from degradation for increased stability.
Among the tested PEG-FGF2 conjugates, compound 6 exhibited the
strongest stability.